This application will establish new pathways required for initiation of lens cell differentiation and will reveal a long-sought after mechanism for lens organelle elimination upon lens cell maturation to achieve lens transparency. We have recently discovered that low-level caspase-3 activation is required for initiation of lens cell differentiaton and that a new macroautophagy pathway initiated by JNK down-regulation directs organelle elimination during lens cell maturation. AIM 1 will establish a novel function for ?-crystallin in regulating mitochondrial cytochrome c release to ensure low-level caspase-3 activation required for lens cell differentiation. AIM 2 will establish how low-level caspase-3 activity transmits its differentiation initiation signal through activation of caspase 3-dependent DNAase (CAD) and Mst1 (and its downstream effector ?H2AX) to direct chromatin remodeling events that we propose are required for lens cell differentiation. AIM 3 will functionally define the macroautophagy proteins and pathways that direct the temporal and spatial removal of organelles during lens cell maturation. The logic, feasibility, and potential success of these AIMs is supported by our strong preliminary data demonstrating that: ?-crystallin translocates to the mitochondria where it complexes with cytochrome c to modulate its release and likely ensure low-level caspase-3 activation required for initiation of lens cell differentiation; CAD is activatd and H2AX phosphorylated and bound to DNA in a caspase-3-dependent manner during initiation of lens cell differentiation likely to initiate DNA strand breaks and ?H2AX DNA binding leading to nucleosome positioning changes required for initiation of lens differentiation- specific gene expression; and key macroautophagy regulatory proteins exhibit expression patterns consistent with their functioning in the JNK-regulated MTORC1 pathway that leads to removal of lens cell organelles upon lens cell maturation. The AIMs proposed are significant because their testing will establish new mechanisms crucial to regulation of the decision of lens cells to begin their differentiation program, as well as developmental processes critical for lens transparency. The work will be applicable towards understanding the differentiation, development and disease states of other tissues since the regulatory molecules examined are common to many other tissues. The proposed AIMs are conceptually innovative since they establish new roles and requirements for classic cell death regulators in normal signaling roles in the cell that regulate differentiation and development. The AIMs are technically innovative since they employ sophisticated lens organ culture methods, gene expression and deletion strategies, signaling assays and chromatin monitoring techniques developed and established in our laboratories.